Fluorochrome conjugated anti mouse antibodies

Model antigen OVA was purchased from Sigma-Aldrich (St. Louis, MO, USA), CpG 5′-TCCATGACGTTCCTGACGTT-3′). FAM-labeled CpG and control ODN (5′-TCCATGAGCTTCCTGAGCTT-3′) were obtained from Sangon (Shanghai, China). Carboxyfluorescein diacetate succinimidyl ester (CFSE) dye and the Micro BCA protein assay kit (BCA) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Fluorochrome-conjugated anti-mouse antibodies were obtained from eBioscience (San Diego, CA, USA) and BioLegend (San Diego, CA, USA). Recombinant mouse GM-CSF and IL-4 were obtained from PeproTech (Rocky Hill, NJ, USA). Cy5-SE was purchased from Fanbo Biochemicals Company (Beijing, China). GSH, ethanol, and NaCl were all of analytical grade. The medium for culturing dendritic cells, splenocyte cells, and tumor cells was RPMI 1640/DMEM (Gibco, Carlsbad, CA, USA) supplemented with 10% (v/v) fetal bovine serum (Gibco, Carlsbad, CA, USA), 100 U/mL penicillin (Invitrogen), and 100 μg/mL streptomycin (Invitrogen).

Leukocytes isolated from peritoneal cavity, spleen and liver tissues were suspended in cold PBS (1 x 10⁷ cells/mL) and incubated with 1 μl/mL anti-mouse CD16/32 (eBioscience, San Diego, CA) for 5 minutes to block non-specific Fc receptor-mediated antibody binding. One million cells were then transferred to polystyrene tubes and incubated with 0.5 μg of fluorochrome-conjugated specific antibodies or isotype control antibodies (4°C, 30 minutes), followed by washing with 2 mL cold PBS and centrifugation (300 x g for 5 minutes). The cell pellet was then resuspended in 250 μL cold PBS. Flow cytometry was performed using BD Accuri C6 instrument (BD Biosciences, San Diego, CA). Data were analyzed using Accuri C6 software. The following fluorochrome conjugated anti-mouse antibodies (eBioscience, San Diego, CA) were used: anti-CD3-FITC, anti-CD4-PerCPCy5.5, anti-CD4-FITC, anti-CD8-PE, anti-CD19-PE, anti-F4/80-FITC, anti-CD49b-APC, anti-CD69-PE, anti-CD69-PerCPCy5.5 and respective isotype controls.